Is IC50 Measuring Cytotoxicity or Growth Inhibition in vitro?

Short answer: The IC50 can measure both; it is context-dependent.  Whether or not the IC50 is reflecting a 50% Cytotoxicity or a 50% Growth Inhibition largely depends on what readout reagent is being utilized.

IC50 is ambiguous because it’s an effect-measurement: the question is, what is the IC50 measuring an effect of?

The most common readout reagents I’ve seen are Celltiter Glo, which measures ATP indicative of cell viability and cellular proliferation, and Caspase 3/7, which measures the apoptotic precursor Caspase 3 and Caspase 7 which are indicative of impending cell death.

These readout reagents vary widely in cost, where Celltiter Glo costs 75% less than Caspase 3/7.  This makes Celltiter Glo a popular readout choice: it effectively measures ATP for cell viability and it saves money compared to purchasing Caspase 3/7.  However, a lack of ATP is not the same as cell death, and a low measurement of ATP is not the same as cell death.

The downside of Celltiter Glo and measuring ATP is that senescent cells, and cells that are locked into a P0 replication phase, do not produce ATP as a proliferating cancer cell would.

This means that cells experiencing growth inhibition will appear as if they have underwent cell death when compared to proliferating cancer cells.  If given enough time, the disparity between the growth inhibited cells and the proliferating cells will generate an IC50.

Viability measurements, such as ATP in Celltiter Glo, rely on the cell’s proliferation and division.  If that division is blocked or the cells are senescent then the measurement may not truly be related to cytotoxicity at all.  The IC50 measurement could be misrepresented as cytotoxicity when in reality it is measuring the proliferation rate.

How can we determine if a drug is cytotoxic or if a drug is growth inhibitory?

Image the cells as they grow (Incucyte), or try a different readout such as Caspase 3/7.

Is ‘Cytotoxicity’ better than ‘Growth Inhibition’?

It depends.  It depends on the cancer type and the cell line being used (not all in vitro assays match the cancer cell line to the intended target cancer: I wrote about that here).  If the target cancer is a solid-tumor type, then cytotoxicity would be paramount to eliminate without a radical surgery being needed.  However, if it’s a fast-growing cancer or tumor, then growth inhibition is important in order to limit the overall progression of the disease.

Why does this matter?

The concern is if the assay is long standing, over multiple days, then the drug (or released payload: see my reasoning here) may be growth inhibitory, and may not be classically cytotoxic at all. This matters because if the higher translatability we can achieve between in lab and in vivo studies that translate into meaningful clinical results then it becomes relevant to helping patients and highlights the importance of lab work being seen from a business level.